Cell Culturing
Cells generally exist as three-dimensional aggregates in the body. However, when cells are cultured in an artificial environment, it is common to use the classical plate culture method in which the cells are cultured two-dimensionally in a manner plated as a monolayer on the bottom of the culturing vessel, or a suspension culture method in which cells are cultured while dispersed in a liquid culture solution. Cells most suited for the plate culture method are cells having relatively high adhesion, but even when such suitable cells are used, differences in the culturing environment can often result in significant changes in the properties of the cells. With suspension culture methods as well, certain cells are suitable while others are not.
With increasing demand for in vivo proteins to be used for medical purposes, such as vaccines, enzymes, hormones, antibodies, cytokines and the like, interest is becoming increasingly focused on mass production of such in vivo proteins by cell culturing. In addition, with ever increasing interest in cell transplantation for regenerative medicine, greater focus is being directed toward methodologies for efficient and convenient culturing of large volumes of cells.
For suspended cells of E. coli and the like, research is being conducted on techniques for mass culturing in large-scale culturing tanks. Mass culturing of suspended cells using large-scale culturing tanks requires large volumes of culture solution and an agitating apparatus. Increasing focus is also being directed toward research in which substances are produced using adherent cells, as research on such cells continues to progress. When it is attempted to perform mass culturing of adherent cells, the cells will only expand two-dimensionally when the classical plate culture method is employed, and therefore a large culturing area is necessary.
A method using a bioreactor or cell culture support has been reported as a method of culturing large volumes of cells in a three-dimensional environment (NPL 1 and PTL 1). Methods using a bioreactor include a method in which a fibrous material such as a glass fiber material is accumulated in a column, and the cells are continuously cultured in the space to produce a substance (PTL 2). Microcarriers, which are microparticles on which cells can adhere and grow, are being widely studied as typical cell culturing supports (PTLs 3 and 4).
PTL 4 mentions viral production as an example, and teaches that, in cell culturing methods using microcarriers, the most important factor for raising production volume and increasing efficiency is to reach a high-density cell culture. Also important is whether the cells can efficiently and conveniently proliferate, and can be transplanted and seeded onto the microcarrier support. In this regard, in a cell culturing system using microcarriers it is necessary to carry out sufficient agitation and diffusion so that the microcarriers do not aggregate together. Since this requires a volume space allowing adequate agitation and diffusion of the culture solution in which the microcarriers are dispersed, there is a limit to the density at which the cells can be cultured. In addition, issues still remain in terms of volume and efficiency because it is necessary to separate the fine particles with a separable filter in order to separate the microcarriers and the culture solution.
Methods of continuous mass culturing of spheroid cells by three-dimensional culturing using methyl cellulose or gellan gum have also been devised as different methods from microcarrier culturing (NPLs 2 and 3), but such methods are not only limited to use with spheroid cells, but they also require complex procedures such as precise monitoring of the state of culturing to obtain fine granular spheroid clumps.
A desire exists to develop and establish a cell culturing method that can culture large numbers of cells by a process that is convenient and automatable.
Bioreactors and microcarrier culturing methods using hollow fiber cultures or cellulose cubes have been widely developed as systems for culturing of adherent cells using a support. The classical methodology, as described in PTL 5, involves continuously feeding a medium that has been aerated with air containing 5% CO2, to aggregates of the cells and a culturing support, to allow continuous culturing to be carried out. Such methods have been difficult, however, because the apparatuses used are complex. At actual field of production, microcarriers are most commonly used as cell culture supports (for example, NPL 4). Methods of prolonged culturing using such microcarriers in combination with a medium supply system also continue to be studied (PTLs 6 and 7, and NPL 5). Even in methods using microcarriers, however, the apparatuses used are often complicated and can present a problem in that the culturing efficiency cannot be adequately improved over biological systems, for example. PTL 8 describes a methodological idea thought to be more efficient, but it does not describe a specific example of actual culturing, and specific materials suited for the methodology are not mentioned. Hence, there is a demand for establishment of a more convenient and efficient continuous cell culturing apparatus.
Porous Polyimide Film
The term “polyimide” is a general term for polymers including imide bonds in the repeating unit. An “aromatic polyimide” is a polymer in which aromatic compounds are directly linked by imide bonds. An aromatic polyimide has an aromatic-aromatic conjugated structure via an imide bond, and therefore has a strong rigid molecular structure, and since the imide bonds provide powerful intermolecular force, it has very high levels of thermal, mechanical and chemical properties.
Porous polyimide films have been utilized in the prior art for filters and low permittivity films, and especially for battery-related purposes, such as fuel cell electrolyte membranes and the like. PTLs 9 to 11 describe porous polyimide films with numerous macro-voids, having excellent permeability for gases and the like, high porosity, excellent smoothness on both surfaces, relatively high strength and, despite high porosity, also excellent resistance against compression stress in the film thickness direction. All of these are porous polyimide films formed via amic acid.